Liposomes are highly advanced assemblages consisting of concentric closed membranes formed by water-insoluble polar lipids, particularly phospholipids. Other substances, such as cholesterol, may also be included in the membrane. Stability, rigidity, and permeability of the liposomes are altered by changes in the phospholipid composition. Membrane fluidity is generally controlled by the composition of the fatty acyl chains of the lipid molecules. The fatty acyl chains can exist in an ordered, rigid state or in a relatively disordered fluid state. Factors affecting rigidity include chain length and degree of saturation of the fatty acyl chains and temperature. Larger chains interact more strongly with each other so fluidity is greater with shorter chains. Saturated chains are more flexible than unsaturated chains. Transition of the membrane from the rigid to the fluid state occurs as the temperature is raised above the "melting temperature". The melting temperature is a function of the length and degree of unsaturation of the fatty acyl chain.
In addition to temperature and phospholipid composition, inclusion of a sterol, such as cholesterol, or a charged amphiphile can alter the stability, rigidity and permeability of the liposome by altering the charge on the surface of the liposome and increasing the distance between the lipid bilayers. Proteins and carbohydrates may be incorporated into the liposomes to further modify their properties.
Liposomes are classically prepared by dissolving an appropriate concentration of phospholipid in an organic solvent, evaporating the solvent, and subsequently disrupting the dry lipid layer with excess water or buffer. Substances can be entrapped within the liposomes during formation. "Entrapment" is defined as either the incorporation of a lipophilic substance into the lipid framework of the bilayer or the passive encapsulation of a water-soluble substance in the aqueous compartments. These substances include proteins such as enzymes, hormones, and globulins, polyamino acids, nucleic acids, drugs, vitamins, and virus. An excellent review of liposomes and substances which have been incorporated into liposomes is "Liposomes" by Gregory Gregoriadis found in Drug Carriers in Biology and Medicine, Chapter 14, 287-341, G. Gregoriadis ed. (Academic Press, N.Y., 1979).
The first studies of in vivo injection of liposomes investigated introduction of enzymes into cells via liposomes. Other studies followed on the transport of various substances into otherwise inaccessible cellular regions, both in vivo and in vitro. In general, the fate of liposomes in vivo is dependent on their size, charge, lipid composition and other physical characteristics. Injected intravenously, larger or negatively charged liposomes are cleared more rapidly than smaller or neutral or positively charged ones. Liver and spleen tissues are primarily responsible for removal of liposomes from the blood and the peritoneal cavity. Following local injection, large liposomes are retained and disintigrated at the site of injection. Small, subcutaneously injected liposomes enter the circulation.
A study by Gregoriadis and Ryman entitled "Lysosomal Localization of Beta-Fructofuranosidase Containing Liposomes Injected Into Rats" in Biochem J. 129, 123-133 (1972) reported in vivo distribution of radioactive beta-fructofuranosidase-containing liposomes over time. Activity was found to decline to 50% of the injected dose within one hour. With six hours much of the activity was recovered in the liver and spleen.
In general, the extent of retention in vivo of substances by liposomes is dependent on the physical characteristics of the substance such as molecular weight or hydrophobic bonding, composition and integrity of the liposomes, and the presence of disruptive blood components. For example, addition of 10 mole % or more of cholesterol into the lipid bilayer may decrease release of an entrapped substance while interaction of some blood components with the liposomes may dramatically increase the rate of release.
Unfortunately, there are a number of disadvantages to using liposomes as an in vivo drug carrier. For example, liposomes are known to act as powerful immunological adjuncts to entrapped antigens and caution must be exercised when enzymes or other proteins of xenogeneic origin are entrapped in the liposomes. The rate of diffusion of the drug is difficult to control. This is a function of the inherent instability of the liposomes as well as the presence of specific blood components which accelerate the diffusion of certain drugs through liposomal bilayers. By their nature, some substances are poorly entrapped in liposomes and diffuse rapidly in circulation. Release of the entrapped substance in "pulses" or at a specified time or in response to a particular stimuli has not yet been possible. Still another problem has been the difficulty of targeting any cells or organ other than the liver or spleen.
It is therefore an object of the present invention to provide a method and system wherein substances are entrapped in liposomes for subsequent in vivo or in vitro release and where the time and duration of release of the entrapped substance are controlled by manipulation of the liposomes.
It is a further object of the present invention to provide such a system wherein the liposomes are protected from destructive forces in their biological environment, such as shearing forces in blood and tissues or the enzymatic action of lipases or other enzymes in tissue or blood, or phagocytosis by macrophages or polymorphonuclear leukocytes.
It is yet another object of the invention to provide a system wherein multiple entrapped substances can be combined and/or sequentially released in response to a specific stimuli or after a predetermined time.